N301 INDUSTRIAL & ENVIRONMENTAL

MICROBIOLOGY.
CHEMOSTAT PROJECT
IMPROVEMNET OF CITRIC ACID PRODUCTION BY
ASPERGILLUS NIGER IN A CONTINUOUS CULTURE.

Barry Cayford (990732B), Karen Sutton (), Michael Bramwell-King (991186J),

Samer Chami (991713J)
Department of Biotechnology, Murdoch University.

The aim of this chemostat was to from which culture liquid is removed at
maximise citric acid production by the same rate (F). The culture volume
Aspergillus niger. Using dissolved (V) is kept constant, and the dilution rate
copper ions in three varied (D) is defined as D=F/V (h-1). The key to
concentrations as the growth the mode of action of the chemostat is
limiting substrate, the citric acid the composition of the medium in the
concentration increased from 24 reservoir. All components in this
mM to 32 mM over the three medium, other than the growth-limiting
trials. As citric acid production substrate, are present in excess. The
increased, a decline in the specific growth rate () of a bacterial
concentration of biomass was culture is defined as:
observed.  = 1/x * dx/dt = d ln x/dt = ln2/td
Where x is organism concentration (mg
Introduction dry weight/ mL) at time t, and t d is the
In a continuous culture fresh medium is time required for the concentration of
introduced continuously into the reactor organisms to double (doubling time).
vessel, culture liquid containing micro (Veldkamp, 1976).
-organisms being concomitantly Although the production of citric acid by
removed at the same rate (Veldkamp, fermentation using Aspergillus Niger has
1976). been a commercial process since 1923,
. The unique properties of continuous there still remain many gaps in our
culture make possible the study of knowledge and understanding of the
physiological and ecological problems, fermentation. Recent reviews indicate
which cannot be tackled with any type of that we don’t know with any certainty
closed culture system. which biochemical pathways are
The most commonly used continuous involved and there is still no unanimity
culture is a single stage flow controlled concerning the optimum physiological
system named a “chemostat”. conditions required to obtain a high yield
A chemostat with commonly used (Smith, 1977).
ancillary equipment is shown Molasses has been mostly used as the
diagrammatically in Fig. 1 Air Up-Lift carbon substrate, while substrates such
Reactor. as starch hydrolysates have also been
Fresh sterile medium is pumped from a used. When crude substrates are used it
medium reservoir into the culture vessel is necessary to reduce the level of iron
and other trace metals, which interfere g/L) the rate of citric acid formation is
with citric acid production. This is proportional to the biomass level:
normally achieved either by precipitation  P/  t = X
with ferricyanide or by the use of a Where  P/  t is the rate of citric acid
cation exchange resin. production (g/l/h); X is the mycelium
Strains of Aspergillus Niger vary concentration (g/L); and  is a metabolic
considerably in their capacity to produce activity of the cells. The changes in the
citric acid and excrete it into the growth citric acid production rate per unit of
medium at high concentration. The biomass, with the different mycelial
amount of citric acid produced can be concentrations resulted from the
increased by induced mutation. inhibition of citric acid synthesis by
Aspergillus Niger can grow in liquid decrease in the level of dissolved oxygen
culture either as loose filamentous in high mycelial densities (Smith, 1977).
mycelium or in a pellet depending on the
growth conditions. Most studies on citric Factors affecting citric acid production
acid fermentation indicate that pellets are:
with a loose structure and a diameter not
exceeding 1 mm provide the best pH: The most favourable hydrogen ion
mycelial form for producing high yields conc. Is between 2.0 and 3.0. The low
of citric acid. Pellet formation is pH is optimal for citric acid production
influenced by many environmental and minimizes the formation of
factors; pH, aeration and agitation, and undesirable organic acids such as
inoculum size appear to be particularly gluconic and oxalic acid. It also reduces
critical in producing a suitable inoculum the risk of contamination by other
for the citric acid fermentation (Smith, organisms.
1977).
Temperature: Temperatures of 28-30oC
There are five successive phases in the have been proposed to obtain high yields
fermentation. The period of spore and rapid rates of accumulation of citric
germination is followed by an acid. At higher temperatures the
exponential growth phase. The fermentation process is very rapid and
maximum value of specific growth rate abundant mycelial growth occurs
in this phase is dependant on causing large amounts of sugars to be
temperature:  max = 0.13 h-1 at 26oC oxidized to CO2 thus lowering the yield
and 0.28 h-1 at 32 oC. of citric acid.
After exponential growth there is a
period of growth disturbance which can Aeration and agitation: Oxygen
be explained by perturbation in the absorption rate decreases with increasing
metabolism of A. niger caused by the amounts of mycelium in culture.
exhaustion of ammonia and phosphate Changes in the specific rate of citric acid
from the medium. This is followed by formation results from the inhibition of
the period of citric acid production, its synthesis caused by a decrease in the
which continues until the available level of dissolved oxygen at high
carbohydrates are exhausted production mycelial densities.
then ceases. A major factor in the
efficient production of citric acid is the Metals and other medium components:
amount of biomass formed. Good citric Small quantities of iron accompanied by
acid producing strains from 10-15 g dry a limited quantity of zinc are essential to
weight of mycelium per litre of medium. obtain a high citric acid yield. Copper
At low mycelium concentrations (up to 4 has been used as an antagonist to iron.
The addition of copper also offers the In this experiment, using an internal loop
additional advantage that is selectively airlift reactor, we aimed to maximise
inhibits the growth of contaminating citric acid by Aspergillus niger using
penicillia. (Smith, 1977). dissolved copper ions as the growth
limiting substrate.
Air Up-lift bioreactors: Materials & Methods
Airlift bioreactors are simple in design
Organism:
and construction. Fig. Having no form
A non-sporulating, prolific slope of
of mechanical agitation and requiring
Aspergillus Niger, grown on a complex
relatively low power inputs per unit
agar incubated at 28oC, was aseptically
volume, they make an economically
inoculated into the chemostat. The
attractive proposition.
reactor was initially run as a batch
The airlift fermenter has been
culture to reach a large biomass
successfully used for citric acid
concentration.
production and many other fermentation
processes. Industrial use of airlift
fermenters is likely to develop in areas Equipment:
where very low shear stresses are  Water bath.
required, good oxygen transfer rates  Heater (water).
have been achieved at a shear stress of  Thermometer.
0.05 Nm-2.  Air source, air depressurising
Two broad groupings of airlift unit, airflow regulator.
fermenters are available:  Media pump.
1. Internal loop airlifts with an internal  2 x 2L Glass bottles.
baffle splitting the vessel and  Drip-feed vessel, syringe x2,
creating a riser and a downcomer needle, stopper.
section.  Electrical source.
2. External loop airlifts with the riser  Tubing.
and the downcomer being separate  Reactor: Air Up-Lift Reactor,
sections connected by horizontal see Fig. 1 Air Up-Lift Reactor.
sections near the top and base of the
bioreactor. All equipment were autoclaved prior to
commencement.
The mixing in such bioreactor types is a
consequence of the different gas hold-
Medium:
ups in the aerated and unaerated zones,
Three trials were used in the experiment.
thus resulting in differing bulk densities,
Each trial was run with the same media
which the fermentation broth to flow.
composition, as indicated in Table 1.
Thus mixing is not as efficient as that
Media Composition.
found in the stirred tank reactors, and
TABLE 1
oxygen transfer rates are consequently
Media Composition.
lower, which may result in lower
Glucose 50 g/L
specific productivities being achieved. NH4NO3 0.8 g/L
Process control is also less effective, as MgSO4.7H2O 0.1 g/L
concentration gradients are more KH2PO4 0.5 g/L
pronounced in this type of bioreactor, (NH4)2SO4Fe(SO4)2.24 H2O 0.0001 g/L
which makes it unsuitable to processes ZnSO4.7 H2O 0.0001 g/L
involving highly viscous fermentation
fluids (Smith, 1994).
The copper concentration was varied in pre-calibrated pH meter in a 10 mL
each trial in order to maximise citric acid sample from the reactor vessel.
production.
TABLE 2 Microscopy:
Varied Copper Concentrations. A slide was prepared from the biomass
CuSO4.5H2O 0.1 g/L 0.25 g/L 0.5 g/Lin the product vessel for each trial. The
morphology of the spores’ hyphae were
The substrate media was prepared examined.
according to Kristiansen and Sinclair.
However, the copper concentrations Residual glucose in the reactor:
were varied according to Longford, Two 1.5mL eppendorf tubes filled with
Table 2. Varied Copper Concentrations. culture from the reactor vessel were
All media was autoclaved prior to spun in a centrifuge at 14000 rpm for
commencement of each trial. 10 minutes. A neat sample was then
prepared by removing 1ml of the
Reactor Conditions: supernatant and transferring it into
The temperature was maintained another 1.5mL eppendorf tube. A 1/10
o
between 28 and 32 C by a heater in the dilution of the supernatant was then
water bath for the duration of the tested for glucose by a glucose analyser
experiment. by the method of gas chromatography.
The airflow rate was set at 25 L/h. At
this rate the Up-Lift vessel maintained Citric acid production:
thorough mixing. 5mL of the outflow material was
The reactor volume was kept at 1500 mL accurately removed from the product
throughout the experiment. vessel and aseptically transferred into a
The substrate flow rate used was 40 100 mL conical flask. One drop of
mL/h. Higher rates resulted in biomass phenol phthalein indicator was added.
washout. The sample was titrated against 0.1 M
The pH of the reactor was kept within NaOH until the first permanent pink
the parameters of 3.3 and 3.6. According colour emerged.
to Kristiansen and Sinclair, this pH (See appendix for sample calculation)
maintained correctly stunted hyphae
length. Biomass:
1mL of the culture was removed from
Sampling: the reactor vessel and placed in a pre-
To ensure that readings were taken at weighed 1.5 mL eppendorf tube and
24hour intervals, sampling was carried spun for 10 minutes at 1400rpm. The
out daily. The chemostat was tested for pellet was resuspended in deionised
pH, citric acid concentration in the water. This sample was spun at
production vessel, residual glucose in the 14000rpm for 10 minutes and the
reactor and Biomass in the reactor. resulting supernatant was discarded. The
pellet was dried in an oven at 110 oC
Temperature: overnight and weighed the next morning.
The temperature was maintained Nett weight is the amount of dry weight
o o
between 28 C - 32 C throughout the per mL of culture.
experiment, measured with the aid of a
thermometer.

pH:
The pH was tested by placing a sterile
Results Biomass Produced in Trials
TABLE 3
3.5
Chemostat Results.
3.0
Trial 1 2 3
2.5
[Cu2+] (g/L) 0.1 0.25 0.5

Biomass (g/L)
2.0
Condition* 1.5
Temp (oC) 29 31 30 1.0
Airflow (L/h) 25 25 25 0.5
Reactor Volume 1500 1500 1500 0.0
(mL) 0 1 2 3 4
Flow rate (mL/h) 40 40 40 Trial
pH 3.4 3.3 3.5
Fig. 3 Biomass Produced in Trials. The
Substrate Volume 1000 1000 1000
Product Volume 1000 1000 1000
biomass reduced as copper concentration
Residual 0.008 0.005 0.003 increased.
[Glucose] (g/L)
Biomass (g/mL) 2.898 2.536 1.987
Titration Residual Glucose in Trials
[NaOH] M 0.1 0.1 0.1 10.0
Sample (mL) 5 5 5
Residual Glucose (ppm)

NaOH (mL) 1.2 1.4 1.7 8.0

Citric Acid (mM) 24 28 32 6.0

* At time of sampling
4.0
Copper Concentration of Trials
2.0
0.6

0.0
Copper Concention (kg/L)

0.5
0 1 2 3 4
0.4 Trial

0.3
Fig. 4 Residual Glucose in Trials. The
0.2
residual glucose concentration reduced
0.1 as copper concentration increased.
0.0 Glucose was ultimately converted to
0 1 2 3 4
citric acid.
Trial

Fig. 2 Copper Concentrations of Trials. Effect of Copper Concentration

The copper concentration was the on Citric Acid Production
variable used to limit biomass growth 35
30
and maximise citric acid production. The
[Citric Acid] (mM)

25
first trial ran at [Cu] 0.1 g/L and this was 20
successively increased to 0.5 g/L. 15
10
5
0
0 0.1 0.2 0.3 0.4 0.5 0.6
[Copper] (ppm)

Fig. 5 Effect of Copper Concentration

on Citric Acid Production. As the copper
concentration increased the citric acid
production rose. The range varied from
24 mM observed in trial one, to 32 mM
at a copper concentration of 0.5 g/L.
 substrate was converted into biomass
Discussion than was utilised for citric acid
The aim of this chemostat was to production. These two processes are
maximise citric acid production by competitive, and only at abnormally low
Aspergillus niger. The citric acid conditions of pH will the organism
concentration increased from 24 mM to produce the product.
32 mM over the three trials. Fig. 5 Effect
of Copper Concentration on Citric Acid Combined with a low pH, relatively high
Production. As citric acid production soluble concentrations of copper affect
increased, a decline in the concentration the rate at which A. niger converts
of biomass was observed. Fig. 3 glucose to citric acid, Crueger et al.
Biomass Produced in Trials. The copper affects aconitase, an enzyme
in the TCA cycle. But this stops the
Initially the experiment was run as a cycle and inhibits ATP production. A.
batch culture to increase biomass to an niger has the ability to use the enzyme
appropriate level for measurable rates of pyruvate decarboxylase and restart the
citric acid production. A. niger has a low TCA cycle. It is this step which allows
td making it appropriate for time limiting for the production of citric acid.
experiments. Using the Uplift reactor we We chose to vary copper concentrations
found that a biomass concentration of and observe the effect this had on citric
approx. 3.0 g/mL was ideal as this gave acid production. In the first trial, 0.1 g/L
quantitative yields of citric acid but did Cu was used. This resulted in 24 nM
not block the reactors flow. However, as citric acid produced. With no cooper in
each trial neared completion, biomass in solution, only 0.02mM citric acid was
the reactor increased and blocked the produced. The second trial used 0.25 g/L
spill over outflow pipe. This increase in Cu and resulted in 28 mM citric aid. The
biomass was most likely due to substrate final trial yielded 32 mM with 0.5 g/L
limitation and the subsequent utilisation Cu. As seen in Fig. 5 Effect of Copper
of citric acid still present in the reactor. Concentration on Citric Acid
As the reactor had little to no free copper Production. The citric acid rose as the
ions in solution, most had flowed out copper concentration in creased. It can
with the product, the residual citric acid be concluded that at higher
was completely utilised for growth, concentrations of copper, the metabolic
Kristiansen and Sinclair. pathways for ATP generation are slowed
and the pathway for citrate synthesis
From observation under a 400x emphasised. But above 0.5 g/L it is not
microscope, the spore morphology was known whether there would be an
that of 0.4-0.7 mm diameter spheres, increase in citric acid production or
white in colour with stunted hyphae, death of the cells as copper
about 0.2 mm in length, and small concentrations in excess of 500 ppm (0.5
nodules around the filaments. According g/L) can result in cell lysis, Milsom et al.
to Kristiansen and Sinclair, this structure
results in optimum citric acid Using the uplift reactor, both aeration
production. This was achieved by and mixing were intrinsically linked.
maintaining a low pH, 3.4, during the The rate of airflow determined the extent
experiment. It was observed that at of mixing (in our basic reactor). It was
higher pH the hyphae grew longer, the found that at an airflow rate of 25 L/h,
nodules were less stunted and there was the mixing in the reactor was adequate
a lower concentration of citric acid for biomass circulation and provided a
produced. Therefore at higher pH, more
low sheer pattern. The low sheer prevent growing into the surrounding water bath
hyphal damage occurring, Smith et al. or into the air, feed or outflow tubes.
It was observed that A. niger has the This can be achieved by having a large
ability to degrade citric acid. The use of distance between the top of the culture
the desired product as a secondary and the top of the reactor, and by
metabolite was difficult to prevent, as maintaining quite high flow rates.
the holding time was high. This was
overcome using a flow rate of 40 mL/h. A similar problem was that of the
This rate prevent washout but allowed growing in the harvest vessel and
sufficient time for the organism to utilise degrading the products, this could be
the substrate, as evident by the glucose easily prevented by using a biomass
utilisation seen in Fig. 4 Residual sieve to stop the biomass from sitting in
Glucose in Trials. One could then draw and changing product levels in the
the conclusion that citric acid production harvest.
by A. niger is best done using a Wide bore tubing should be used so that
chemostat, not a batch reactor. chance of blockage occurring is
minimised
With the aid of Darent Longford and
Ralf Cord-Ruwisch, an uplift reactor was Use minimum amount of glucose to
created in order to overcome some of the prevent excess biomass production,
problems encountered using a stirred minimal glucose also helps give optimal
reactor. citric acid production.
The stirred reactor kept clogging with
overgrown biomass. The pump we had Use clear reactor and water bath vessels
gave two different flow rates for feed in as this allows for the levels of both the
and product out, resulting in a culture and biomass to be monitored
fluctuating reactor volume. And the easily.
product out tubing kept blocking with
extracted biomass. Instead of making one stand medium
The uplift reactor solved these problems. make up separate standards of each
Stirring was controlled using the airflow nutrient, as this allows for easy variation
rate. One pump was used for substrate in quantities and also more accurate
feed in only. The outflow was a spillover concentrations, especially when using
design and wouldn’t clog. Consequently the trace amounts in the micronutrients
the reactor volume was precisely
maintained. The transparent reactor
vessel allowed for qualitative estimates
of copper utilisation and the simple
design proved cheap in comparison to
the stirred reactor.

Suggestions
Air regulator must be used, to prevent
fluctuations in airflow.

ICP measurements for residual copper in

harvest vessel.

Reactor design needs to be so that the

growing culture is prevented from
Appendix Legisa M. and Gradisnik-Grapulin M.
(1995) Sudden Substrate Dilution
*Sample Calculation: Induces a Higher Rate of Citric Acid
Citric acid production: Production by Aspergillus niger. Applied
Titration; Trial 1 and Environmental Microbiology LXI:
[NaOH] = 0.1M = 100 mM (C1) 2732-2737.
Volume of NaOH used = 1.2mL (V1)
Volume of citric acid sample = 5mL Milsom P. and Meers J. (1985) Citric
(V2) Acid John and Sturge Ltd. Selby, New
C1V1 = C2V2 Yorkshire, United Kingdom.
100 * 1.2 = C2 * 5
C2 = [citric acid] = 100 * 1.2/ 5 = 24mM Smith J.E., Berry D.R. and Kristiansen
B. (1980) Fungal Biotechnology.
Academic Press, London, United
Kingdom
References:
Veldkamp H., (1976), Continuous
Cord-Ruwisch R. (2000) Industrial and culture in microbial physiology and
Environmental Microbiology ecology, Meadowfield Press Ltd.,
Laboratory Manual and Lecture Durham, England.
Guide. Murdoch University Press,
Murdoch University, Western
Australia.

Crueger W. and Crueger A. (1984)

Biotechnology; A Textbook of Industrial
Microbiology. Science Tech Inc, USA.

Grewel H.S. and Kalra K.L. (1995)

Fungal Production of Citric Acid.
Biotechnology Advances XIII(2): 209-
234.

Hawksworth D.L. and Kirsop B.E.

(1988) Living Resources for
Biotechnology – Filamentous Fungi.
Cambridge University Press, Cambridge
University, United Kingdom.

Kristiansen B. and Sinclair C.G. (1978)

Production of Citric Acid in Batch
Culture. Biotechnology and
Bioengineering XX: 1711-1722.

Kristiansen B. and Sinclair C.G. (1979)

Production of Citric Acid in Continuous
Culture. Biotechnology and
Bioengineering XXI: 297-315.